Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells

ABSTRACT

The present invention is concerned with a composition and in vitro method for generating a desired cell type and/or tissue type from hair follicular stem cells. The composition and in vitro method are particularly suitable for generating an autologous desired cell type and/or tissue type. Furthermore, the composition and method are especially efficient and suitable for use in the context of cosmetic cell and/or tissue transplantation in recipient areas of a subject experiencing cell and/or tissue loss caused by, for example, a wound, scar, burn injury, tissue degeneration, and aging. The composition and in vitro method are also suitable to circumvent complications related to infections and/or immune rejection of a cosmetic cell and/or tissue implant or graft.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/NL2014/050062 filed Feb. 3, 2014, which claims the benefit ofNetherlands Application No. NL 2010222, filed Feb. 1, 2013, the contentsof which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is in the fields of tissue engineering withapplications in the fields of cosmetic and aesthetic procedures andregenerative medicine. The present invention provides an improvedcomposition and in vitro method for generating a desired cell typeand/or tissue type from at least one hair follicular stem cell. Theimproved composition and in vitro method of the present invention areparticularly suitable for cosmetic or therapeutic cell and/or tissuetransplantation in recipient areas of a subject experiencing cell and/ortissue loss caused by a wound, scar, burn injury, tissue degeneration,diseases and/or aging. The composition and in vitro method of thepresent invention are also particularly suitable to circumventcomplications related to infections and/or immune rejection of acosmetic or therapeutic cell and/or tissue implant or graft in asubject.

BACKGROUND OF THE INVENTION

Interest in pluripotent stem cells has increased dramatically in thelast decade, particularly with regards to their role in the rapidlyexpanding fields of tissue engineering, regenerative medicine, andfacial as well as body rejuvenation technology. Natural pluripotent stemcells are cells endowed with the potential to differentiate into anyfoetal or adult cell types. The procurement of such cells is, however,highly limited and constantly subjected to severe ethical concerns sincenatural pluripotent stem cells are from embryonic origin and thus canonly be obtained from an embryo. Therefore, intensive research effortshave been devoted to uncover alternative non-embryonic sources ofpluripotent stem cells that are free of ethical concerns.

A breakthrough discovery revealed that non-embryonic stem cells (alsoreferred to as adult stem cells or somatic stem cells) exist in almostall adult tissues of the body. Examples of non-embryonic stem cellsinclude, mesenchymal stem cells, hematopoietic stem cells, epithelialstem cells, dermal stem cells, and neural stem cells, hair follicularstem cells, and others. In contrast to embryonic stem cells,non-embryonic stem cells are multipotent rather than pluripotent.Multipotent stem cells have the potential to give rise to cells frommultiple lineages, but not all lineages. In other words, non-embryonicstem cells cannot naturally differentiate into any type of cells, atleast not without undergoing artificial manipulations. Indeed, it wasdiscovered that multipotent non-embryonic stem cells can be artificiallyinduced to become pluripotent in laboratory settings by inducing theforced expression of certain genes. Such artificially transformed cellsare also referred to as “induced pluripotent stem cell”. However, theuse of induced pluripotent stem cells is controversial since such cellsare often highly tumorigenic.

Another breakthrough discovery revealed that not only hair follicularstem cells appear to escape the tumorigenic fate but they also display ahigh proliferative and clonigenic ability while having the potential todifferentiate, however, under artificial conditions, in a variety ofdifferent tissues including neural tissue, eye tissue (e.g. retinalpigment epithelium tissue), heart tissue (e.g. cardiomyocytes), toothtissue, adipogenic tissue, chondrogenic tissue, tissue, and myogeniclineages similar to bone marrow, and others. Therefore, because of theirbroad regenerative potential, great accessibility, and non-oncogenicquality, hair follicular stem cells are currently considered one of themost promising sources of non-embryonic stem cells for the purpose oftissue engineering, tissue transplantation technology, facial and bodyrejuvenation technology, and other cosmetic technologies as well astherapeutic procedures (e.g. regenerative medicine).

Another rapidly developing field is the field of “autologous products”and related cosmetic treatments and therapeutic procedures, which isbased on the principle of taking a subject's own proteins, cells, ortissues and reintroduce them back into the same subject. Such technologyis particularly advantageous in the fields of cosmetic tissueengineering, tissue transplantation technology and facial and bodyrejuvenation technology, therapeutic procedures (e.g. regenerativemedicine) and others, since it prevents the occurrence of complicationsrelated to immune rejection and/or infection. Therefore, the interest inhair follicular stem cells, especially from autologous sources, iscurrently heightened, particularly for developing improved methods forefficiently manipulating hair follicular stem cells in vitro.Specifically, because the number of hair follicular stem cells that canbe harvested from donor hairs is relatively little compared to the largeamount of hair follicular stem cells needed, for instance, in cosmetictissue engineering, tissue transplantation technology and facial andbody rejuvenation technology, therapeutic procedures (e.g. regenerativemedicine) and others, there is a growing need for improved in vitromethods for culturing and expanding (multiplying their number orboosting proliferation) hair follicular stem cells. In parallel, thereis also a great need for improved in vitro methods and compositions forgenerating desired cell types and/or tissue types from hair follicularstem cells, particularly those issued from in vitro culture. Moreprecisely, there is great need for improved cell-specific ortissue-specific compositions, in particular autologous cell-specific ortissue-specific compositions, that enable hair follicular stem cells toproliferate and differentiate in a desired cell type and/or tissue typein a more efficient manner.

Generally, differentiation media for mesenchymal stem cells such as hairfollicle stem cells comprise Foetal Bovine Serum. Serum is a majorsource of viral contaminants which, once present, are difficult toremove from cultures. It can contain viruses, prions and mycoplasma,which may skew the outcome of scientific experiments and may transferdiseases to cultured cell. Additionally, considerable ethical debatesurrounds the production of Foetal Bovine Serum. In the art there is aneed for proliferation and differentiation media devoid of Foetal BovineSerum.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a composition forgenerating a desired cell type and/or tissue type from at least one hairfollicular stem cell, said composition comprising at least oneanti-apoptotic compound, wherein said anti-apoptotic agent is aphysiologically acceptable vanadium compound, at least one anti-oxidantcompound, at least one stem cell enhancer compound, at least oneextracellular matrix compound, and at least one differentiation inducingfactor.

The physiologically acceptable vanadium compound may be selected fromthe group consisting of bis (maltolato) oxovanadium, oxovanadium, andorthovanadium, and is preferably bis (maltolato) oxovanadium.

One or more second or further anti-apoptotic compounds selected from thegroup of a triiodothyronine, estradiol, progesterone, tissue extract,insulin, transferrin, selenium, L-cysteine, adenosinetriphosphate-magnesium chloride, and L-leucine may further be includedin the composition taught herein.

The tissue extract may be bovine pituitary extract.

In an embodiment, the anti-oxidant compound is selected from the groupof quercetin, monohydroxyethyl rutoside, vitamin C, lipoic acid,deferoxamine mesylate, and vitamin E, preferably from quercetin andmono-hydroxyethyl rutoside.

In an embodiment, the stem cell enhancer compound is selected from thegroup consisting of erythropoietin, CD34-positive cell, and retinoicacid, preferably from the group of erythropoietin and CD34-positivecell. In a suitable embodiment, the stem cell enhancer compound iserythropoietin.

The erythropoietin and/or the CD34-positive cell may be derived fromperipheral blood or bone marrow of a donor subject. The CD34-positivecell is obtainable or obtained from a CD34-positive cell line, e.g., ahuman CD34-positive cell line.

The composition taught herein may further comprise at least onedegranulating agent, e.g., Compound 48/80.

The composition taught herein may further comprise an inorganic salt,such as CaCl₂.

In an embodiment, the extracellular matrix compound is selected from thegroup of: platelet rich plasma, laminin, collagen IV, heparin sulfate,entactin, and chondroitin sulfate.

In an embodiment, the extracellular matrix compound is platelet richplasma, which may be derived from peripheral blood of a donor subject.

The composition taught herein may further comprise one or moreingredients selected from the group of albumin, essential andnon-essential amino acids, vitamins, trace elements, organicconstituents, growth supplement, and antibiotics.

The desired cell type may be selected from the group of nerve cell,photoreceptor cell, cardiomyocyte, odontoblast, epithelial cell,keratinocyte cell, fibroblast cell, fat cell, blood cell, immune cell,muscle cell, skin cell, hair follicular cell, osteoblast, osteocyte,osteoclast, and chondrocyte.

The desired tissue type may be selected from the group of neural tissue,eye tissue, heart tissue, tooth tissue, epithelial tissue, keratinocytetissue, fibroblast tissue, connective tissue, fat tissue, muscle tissue,skin tissue, hair tissue, bone tissue, and cartilage tissue.

The differentiation inducing factor may be selected from the groupconsisting of basic fibroblast growth factor, insulin-growth factor,epidermal growth factor, transforming growth factor, nerve growthfactor, fibroblast growth factor, epithelial growth factor, taurin,activin A, and 5-azacytidine.

The insulin-like growth factor may selected from the group consisting ofinsulin-like growth factor type 1 and insulin-like growth factor type 2.

The differentiation inducing factor may epidermal growth factor, andsaid desired cell type and/or tissue type is epidermal cell and/ortissue type, skin cell and/or tissue type, blood vessel cell and/ortissue type, or bone cell and/or tissue type. The epidermal growthfactor may be selected from the group consisting of: heparin-bindingepidermal growth-like growth factor, epiregulin, epigen, betacellulin,neuregulin-1, neuregulin-2, neuregulin-3, and neuregulin-4.

The differentiation inducing factor may be transforming growth factor,and said desired cell type and/or tissue type is cartilage cell and/ortissue type, heart valve cell and/or tissue type, and skeletal musclecell and/or tissue type. The transforming growth factor may be selectedfrom the group consisting of: transforming growth factor alpha andtransforming growth factor beta.

The differentiation inducing factor is selected from activin A, taurin,and epidermal growth factor, and said desired cell type and/or tissuetype is photoreceptor cell and/or retinal tissue.

The differentiation inducing factor may be fibroblast growth factor, andsaid desired cell type and/or tissue type is fibroblast cell, skin celland/or skin tissue, or blood vessel cell and/or blood vessel tissue.

The differentiation inducing factor may be epithelial growth factor, andsaid desired cell type and/or tissue type is keratinocyte cell and/orkeratinocyte tissue.

The composition taught herein may further comprise a serum free growthmedium.

In another aspect, the present invention provides an in vitro method forgenerating a desired cell type and/or tissue type from at least one hairfollicular stem cell, said method comprising the steps of:

-   -   providing at least one hair follicular stem cell;    -   culturing the at least one hair follicular stem cell in a medium        so as to obtain a population of hair follicular stem cells;    -   contacting the population of hair follicular stem cells of        step (b) with a composition taught herein and allowing said        population of hair follicular stem cells to differentiate into        the desired cell type and/or tissue type under conditions        favorable for differentiation; and    -   harvesting the desired cell type and/or tissue type.

The at least one hair follicular stem cell of step (a) may be obtainedfrom at least a part of a hair follicle in the anagen phase. The atleast a part of a hair follicle in the anagen phase may have beenobtained by plucking a hair from a donor area of a subject.

The at least a part of a hair follicle may be contacted with a mediumcomprising collagenase IV.

The hair follicular stem cell is preferably a human hair follicular stemcell.

The medium is preferably a serum-free growth medium, optionally furthersupplemented with at least one tissue extract, e.g., a bovine pituitaryextract compound.

The desired cell type and/or tissue type is preferably intended forintroduction in a recipient region in a subject, and wherein the atleast one hair follicular stem cell of step (a) is derived from saidsubject, and wherein the composition of step (c) comprises at least oneCD34-positive cell derived from said subject and/or platelet rich plasmaderived from said subject.

In another aspect, the present invention relates to use of a desiredcell type and/or tissue type obtainable or obtained by the in vitromethod taught herein for a cosmetic purpose.

The invention also relates to use of a desired cell type and/or tissuetype obtainable by the in vitro method taught herein for cosmetic tissuerepair and/or wound healing.

The invention further pertains to use of a desired cell type and/ortissue type obtainable by the in vitro method taught herein for cosmeticfacial and body rejuvenation.

The invention further provides use of a desired cell type and/or tissuetype obtainable by the in vitro method taught herein for regenerativemedicine.

Finally, the present invention relates to a kit comprising thecomposition taught herein, optionally with a leaflet comprising writteninformation on how to use the composition taught herein to generate adesired cell and/or tissue type.

DETAILED DESCRIPTION OF THE INVENTION General Definitions

The term ‘anti-apoptotic compound’ as used herein refers to a compoundcapable of preventing or reducing apoptosis. The term ‘anti-apoptoticcompound’ is commonly used by the skilled person to describe compoundsor substances having anti-apoptotic activity. Non-limiting examples ofanti-apoptotic compounds include: physiologically acceptable vanadiumcompounds (e.g. bis (maltolato) oxovanadium, oxovanadium,orthovanadium), triodothyronine, estradiol, progesterone, insulin,transferrin, selenium, L-cysteine, L-leucine, adenosinetriphosphate-magnesium chloride, tissue extract (e.g. bovine pituitaryextract), and others.

The term ‘anti-oxidant compound’ as used herein refers to a compoundcapable of inhibiting or reducing the oxidation of other moleculesand/or counteracting the effects of oxidative stress in a cell and/ortissue. The skilled person is well acquainted with the meaning of theterm ‘anti-oxidant’ and recognizes it as well-known category ofcompounds used in various compositions or products. Non-limitingexamples of anti-oxidant include: quercetin, monohydroxyethyl rutoside,vitamin C, lipoic acid, deferoxamine mesylate, vitamin E, and others.

The term ‘stem-cell enhancer compound’ as used herein, refers to acompound capable of enhancing the reprogramming of a stem cell ordifferentiation of a stem cell into a desired tissue type. Stem-cellenhancer compounds are also commonly used to promote expansion of stemcells while maintaining their genomic stability and viability underculture conditions. The skilled person is well acquainted with the term‘stem-cell enhancer compound’ particularly in the context ofregenerative medicine and cosmetic procedures, where stem cells are usedto generate various types of tissues in vitro. Non-limiting examples ofstem-cell enhancer compounds include erythropoietin, CD34-positivecells, retinoic acid and others.

The term “CD34-positive cell” refers to a cell positive for themolecular marker CD34, which is a surface glycoprotein functioning as acell-cell adhesion factor. CD34 may also mediate the attachment of stemcells to bone marrow extracellular matrix or directly to stromal cells.CD34 is also the name for the human gene that encodes the protein. CD34represents a well-known marker for primitive blood- and bonemarrow-derived progenitor cells, especially for hematopoietic andendothelial progenitors. CD34-positive cells may be isolated fromperipheral blood or bone marrow in a subject. It is particularlyadvantageous to isolate CD34-positive cells from peripheral blood orbone marrow of a patient in the context where an autologous tissue graftis desired, for instance. CD34-positive cells is one example of a stemcell enhancer compound.

The term ‘extracellular matrix compound’ as used herein refers to acompound capable of providing structural and biochemical support to thecells in a tissue or agglomerate of cells in in vivo and in vitro (e.g.culture) contexts. Extracellular matrix compounds are also widely knownto promote cell adhesion, cell-to-cell communication and differentiationwithin a given tissue or agglomerate of cells in a culture medium. Theskilled person is familiar with the term ‘extracellular matrix compound’and recognizes that the term ‘extracellular matrix compound’ encompassesa wide variety of compounds useful in the context of cellular or tissueculture. Non-limiting examples of extracellular matrix compounds includeplatelet rich plasma, laminin, collagene IV, heparine sulphate,entactin, chondroitin sulphate, and the likes.

The term “differentiation inducing factor” as used herein refers to acompound that is capable of inducing differentiation of a mesenchymalstem cell, such as a hair follicular stem cell, into a different,desired, cell type, such as neural cell, photoreceptor cell,cardiomyocytes, smooth muscle cells, epithelial cells, and the like. Adifferentiation inducing factor may be any type of compound. Forexample, a differentiation inducing factor may be a well-known growthfactors or any other chemical compound. In order to inducedifferentiation, differentiation inducing factors may be used incombination.

The term “growth factor” as used herein refers to a naturally occurringsubstance capable of stimulating cellular growth, proliferation,cellular differentiation, and/or cellular maturation. Growth factorsexist in the form of either proteins or steroid hormones. Growth factorsare important for regulating a variety of cellular processes. Growthfactors typically act as signaling molecules between cells. However,their ability to promote cellular growth, proliferation, cellulardifferentiation, and cellular maturation varies between growth factors.A non-limiting list of examples of growth factors includes: basicfibroblast growth factor. adrenomedullin, angiopoietin, autocrinemotility factor, bone morphogenetic proteins, brain-derived neurotrophicfactor, epidermal growth factor, epithelial growth factor, fibroblastgrowth factor, glial cell line-derived neurotrophic factor, granulocytecolony-stimulating factor, granulocyte macrophage colony-stimulatingfactor, growth differentiation factor-9, hepatocyte growth factor,hepatoma-derived growth factor, insulin growth factor, insulin-likegrowth factor, migration-stimulating factor, myostatin, nerve growthfactor, and other neurotrophins, platelet-derived growth factor,transforming growth factor alpha, transforming growth factor beta,tumor-necrosis-factor-alpha, vascular endothelial growth factor,placental growth factor, fetal bovine somatotrophin, and cytokines (e.g.IL-1-cofactor for IL-3 and IL-6, IL-2-t-cell growth factor, IL-3, IL-4,IL-5, IL-6, and IL-7).

As used herein, the term “hair follicular stem cells” may include hairfollicle epithelial stem cells, hair follicle mesenchymal-like stemcells, hair follicle melanocyte stem cells, and/or nestin-positive stemcells.

The terms “neural cells and/or neural tissue” and “nerve cell and/ornerve tissue” as used herein refer to cell and/or tissue having originfrom the main component of the two parts of the nervous system; thebrain and spinal cord of the central nervous system (CNS), and thebranching peripheral nerves of the peripheral nervous system (PNS). Theterm “nervous cell and/or tissue” is also commonly used as an equivalentto “neural cells and/or neural tissue” and “nerve cell and/or nervetissue”. In the present invention, these terms are used interchangeably.

The terms “autologous” in the context of the present invention means“derived or transferred from the same individual's body”, and may referto organs, tissues, cells, fluids, or proteins a subject, and which areto be administered to or transplanted in (optionally, another part ofthe body) the same individual. Organs, tissue, cells, or proteinstransplanted by such “autologous” procedure is referred to as anautograft or autotransplant.

The term “allogeneic” as used herein, refers to organs, tissues, cells,fluids or proteins taken from a donor subject, and which are to beadministered to or transplanted in a recipient subject of the samespecies that is genetically non-identical to the donor subject. Organs,tissue, cells, fluids or proteins transplanted by such “allogeneic”procedure is referred to as an allograft or allotransplant.

The term “hair follicle” refers to a mammalian skin organ that produceshair. More particularly, the hair follicle is composed of the dermalpapilla (DP), dermal sheath (DS), outer root sheath (ORS), inner rootsheath (IRS), and hair shaft. The upper and lower parts of the hairfollicle both comprise hair follicular stem cells that are capable ofgenerating a new hair. These are referred to as ‘bulge stem cells’ and‘matrix stem cells’, respectively.

The term “at least a part of a hair follicle” as used herein refers to apart of a hair follicle or an entire hair follicle. The at least a partof a hair follicle comprises at least one hair follicular stem cell, butpreferably more than one. The hair follicular stem cells may be eitherbulge stem cells or matrix stem cells, or both. Preferably, the hairfollicular stem cells comprise at least bulge stem cells or derivativesthereof.

The terms “anagen phase”, “catagen phase”, and “telogen phase” representthe three phases of the natural growth cycle of the hair, including thegrowth phase, the transitional phase (also referred to as involuting orregressing phase), and the death phase (also referred to as resting orquiescent phase), respectively.

The term “three dimensional culture” refers to a method of culturingcells wherein cells are implanted or seeded into an artificial structurecapable of supporting three-dimensional tissue formation. Thesestructures, typically called scaffolds, are critical, both ex vivo aswell as in vivo, to recapitulating the in vivo milieu and allowing cellsto influence their own microenvironments.

The term “expanding” or the term “multiplying” as used herein refer tothe proliferation of one cell to two cells, and so on. During theprocess of hair follicular stem cell expansion or multiplication, a hairfollicular stem cell is self-renewing. Self-renewal of a cell is theability of a given cell to go through numerous cycles of cell divisionwhile maintaining an undifferentiated state, i.e. the hair follicularstem cell remains a hair follicular stem cell regardless of the numberof cell division cycles.

Compositions of the Invention

In a first aspect, the present invention relates to a composition thatis suitable for generating a desired cell type and/or tissue type fromat least one hair follicular stem cell, i.e., for differentiating saidhair follicular stem cell into a different cell type, said compositioncomprising at least one anti-apoptotic compound, wherein saidanti-apoptotic agent is a physiologically acceptable vanadium compound,at least one anti-oxidant compound, at least one stem cell enhancercompound, at least one extracellular matrix compound, and at least onedifferentiation inducing factor. The composition taught herein may bedevoid of the Foetal Bovine Serum (FBS) that is commonly used inculturing and differentiating of cells and is therefore suitable forculture of cells and tissues that are intended to be re-applied into oronto the human body.

Anti-apoptotic compounds, anti-oxidant compounds, stem cell enhancercompounds, extracellular matrix compounds, and differentiation inducingfactors are well-known in the art and may even be commerciallyavailable. Any commercially available anti-apoptotic compounds,anti-oxidant compounds, stem cell enhancer compounds, extracellularmatrix compounds, and differentiation inducing factors may be used forpreparing the composition of the present invention.

In an embodiment, the physiologically acceptable vanadium compound isselected from the group consisting of bis (maltolato) oxovanadium (Casno. 38213-69-3), oxovanadium, orthovanadium and derivatives thereof. Ina more preferred embodiment, the physiologically acceptable vanadiumcompound is Bis (maltolato) oxovanadium. Any physiologically acceptableconcentrations (mg/ml) of Bis (maltolato) oxovanadium can be used in thecomposition of the present invention. However, a concentration of0.01-100 mg/ml, more preferably 0.1-10 mg/ml, more preferably 0.5-8mg/ml, more preferably 0.6-6 mg/ml, more preferably 0.7-3 mg/ml, 0.8-2mg/ml, more preferably 0.9-1.5 mg/ml, more preferably about 1 mg/ml ofBis (maltolato) oxovanadium is preferred in the present invention. Thepresent inventor has found that the use of an anti-apoptotic compoundsuch as a physiologically acceptable vanadium compound, especially Bis(maltolato) oxovanadium, was particularly effective relative to othertypes of anti-apoptotic agents. Specifically, it was found that theincorporation of a physiologically acceptable vanadium compound,especially Bis (maltolato) oxovanadium, into the composition greatlyboosted the efficiency of other ingredients present in the compositionsof the present invention such as growth factors.

In one embodiment, Bis (maltolato) oxovanadium, oxovanadium andorthovanadium may be used interchangeably.

In one embodiment, the composition as taught herein may further comprisea second or further anti-apoptotic agents selected from the groupconsisting of triodothyronine, estradiol, progesterone, insulin,transferrin, selenium, L-cysteine, L-leucine, adenosinetriphosphate-magnesium chloride, and tissue extract.

Tissue extracts are well-known in the art and are commerciallyavailable. Methods and protocols for preparing tissue extracts are alsowell-known in the art. Any tissue commercially available or home-madeextracts can be used to prepare the composition of the invention butbovine pituitary extract is preferred. It may be particularlyadvantageous to incorporate a tissue extract, particularly a bovinepituitary extract, into the composition of the invention for the purposeof maintaining the hair follicular stem cells in an undifferentiatedstate. The maintenance of hair follicular stem cells into anundifferentiated state is important for instance during the culture andexpansion (or multiplication) phases, so that the hair follicular stemcells can undergo self-renewal, i.e. go through numerous cycles of celldivision, while maintaining an undifferentiated state (maintain geneticstability). This process is typically performed to increase thepopulation of hair follicular stems prior to using said population ofhair follicular stem cells for generating a desired cell type and/ortissue type.

In an embodiment, at least two, three, four, five, six, seven, eight,nine, ten, or more second or further anti-apoptotic compounds selectedfrom the group of: triodothyronine, estradiol, progesterone, tissueextract (e.g. bovine pituitary extract), insulin, transferrin, selenium,L-cysteine, adenosine triphosphate-magnesium chloride, and L-leucine arepresent in the composition of the invention. Specifically it was foundby the present inventor that the incorporation of at least two,preferably three, preferably four, preferably five, preferably six,preferably seven, preferably height, preferably nine, and morepreferably ten, was particularly advantageous since it further improvedthe efficiency of the composition of the invention, relative to acomposition where only the physiologically acceptable vanadium compoundis present.

In an embodiment, the anti-oxidant compound of the composition of theinvention is selected from the group of quercetin, monohydroxyethylrutoside, vitamin C, deferoxamine mesylate, vitamin E, and lipoic acid,and a derivative thereof. Anti-oxidant compounds are well-known in theart and are commercially available. Any commercially availableanti-oxidant compounds can be used in the present invention. However, inan embodiment, the incorporation of a flavonoid such as quercetin ormono-hydroxyethyl rutoside, particularly mono-hydroxyethyl rutoside, ispreferred. Any physiologically acceptable concentrations (mg/ml) ofmono-hydroxyethyl rutoside that have anti-oxidant activity can be usedin the composition of the present invention. For example, theconcentration may be in the range of 0.0025-25 mg/ml, more preferably0.025-2.5 mg/ml, more preferably 0.12-2 mg/ml, more preferably 0.15-1.5mg/ml, more preferably 0.18-1 mg/ml, more preferably 0.2-0.5 mg/ml, morepreferably 0.21-0.4 mg/ml, more preferably 0.23-0.3 mg/ml, morepreferably 0.23-0.28 mg/ml, and more preferably is about 0.25 mg/ml ofmono-hydroxyethyl rutoside.

In another embodiment, at least two, three, or more anti-oxidantcompounds selected from the group of: quercetin, monohydroxyethylrutoside, vitamin C, deferoxamine mesylate, vitamin E, and lipoic acidare incorporated in the composition of the invention. It was found bythe inventor that the presence of more than two anti-oxidant compounds,preferably three or more anti-oxidant compounds, further improved theeffectiveness of the composition of the invention, relative to acomposition where only one anti-oxidant compound is present.

In an embodiment, the at least one stem cell enhancer compound of thecomposition of the invention is selected from the group oferythropoietin, CD34-positive cell, and retinoic acid. In an embodiment,the stem cell enhancer is selected from the group consisting oferythropoietin and CD34-positive cell. Particularly erythropoietin wasfound to be very effective.

In another preferred embodiment, the stem cell enhancer compound is aCD34-positive cell. Any physiologically acceptable amounts (cells/nil)of CD34-positive cells can be used in the present invention but anamount of about 1×10¹-1×10⁵ cells/ml, more preferably about 1×10²-1×10⁴cells/ml, more preferably about 5×10²-1×10⁴ cells/ml, more preferablyabout 5×10²-0.5×10⁴ cells/ml, more preferably about 6×10²-0.4×10⁴cells/ml, more preferably about 7×10²-0.3×10⁴ cells/ml, more preferablyabout 8×10²-0.2×10⁴ cells/ml, more preferably about 9×10²-0.15×10⁴cells/ml, more preferably about 1×10³ CD34-positive cells/ml ispreferred. In an embodiment, the CD34-positive cell is obtainable orobtained from a CD34-positive cell line. CD34-positive cell lines areknown in the art and are commercially available, for instance KG-la,KG-1, and NIH3T3. In an embodiment, the CD34-positive cell is obtainableor obtained from a human CD34-positive cell line, which are alsocommercially available such as KG-la, KG-1 and NIH3T3. However, in afurther preferred embodiment, the CD34-positive cell is derived from theperipheral blood or from the bone marrow of a donor subject. Examples ofa donor subject include a non-human mammal subject or a human subject.In a further preferred embodiment, the donor subject is a human subject.Methods for isolating and identifying CD34-positive cells from theperipheral blood or bone marrow are known in the art. For instance,immunohistochemical methods and kits comprising antibodies directedagainst the CD34 antigen can be used to identify CD34-positive cells.

In a more preferred embodiment, the stem cell enhancer compound iserythropoietin. Any physiologically acceptable amounts of erythropoietincan be used in the present invention but an amount of about 0.1 unit to10 units of erythropoietin/ml is used, preferably about 0.3 unit to 8units of erythropoietin/ml is used, preferably about 0.5 unit to 6 unitsof erythropoietin/ml is used, about 0.7 unit to 4 units oferythropoietin/ml is used, about 0.9 unit to 2 units oferythropoietin/ml is used, about 0.95 unit to 1.5 units oferythropoietin/ml is used, more preferably about 1 unit oferythropoietin/ml is used. In an embodiment, erythropoietin may beobtained from a commercial source. In a preferred embodiment,erythropoietin may be obtained from a donor subject, for instance fromthe blood circulation or from bone marrow of a donor subject. Examplesof a donor subject include a non-human mammal subject or a humansubject. In a further preferred embodiment, the donor subject is a humansubject. Methods for obtaining erythropoietin from the peripheral bloodor bone marrow are known in the art. The person skilled in the art iswell acquainted with the term “Unit” (abbreviated as “U”) as a measureunit for amount of erythropoietin. Erythropoietin amounts are expressedin units (U) rather than in grams or moles, because nativeerythropoietin and recombinant human erythropoietin are mixtures ofisoforms with differing bioactivities. In the present invention, oneunit is defined as the amount of erythropoietin that is required toproduce equivalent ³[H]-thymidine incorporation into spleen cells fromphenylhydrazine treated mice to that expressed of 1 unit of theWHO-erythropoietin reference standard (2^(nd) First InternationalReference Preparation).

In an embodiment, at least two, three or more stem cell enhancercompounds selected from the group of erythropoietin, CD34-positive cell,and retinoic acid, are present in the composition of the invention. Itwas found by the present inventor that the presence of at least two stemcell enhancer compounds, preferably the presence of three stem cellenhancer compounds, selected from the group of: erythropoietin,CD34-positive cell, and retinoic acid, led to an increase in theefficiency of the composition of the invention, relative to acomposition wherein only one stem cell enhancer compound is present.

In a further embodiment, the composition of the invention may compriseat least one degranulating agent. In an embodiment the degranulatingagent is Compound 48/80. Degranulating agents, such as Compound 48/80,are known in the art and are commercially available. In anotherembodiment, the composition of the invention may also comprise at leastone inorganic salt. Said inorganic salt may aid in optimizing the actionof the degranulating agent. Inorganic salts are known in the art and arealso commercially available. In an embodiment the inorganic salt isCaCl₂. It may be particularly advantageous to incorporate at least onedegranulating agent such as Compound 48/80 and at least one inorganicsalt such CaCl₂ in a composition of the invention comprising aCD34-positive cell.

In an embodiment, the at least one extracellular matrix compound isselected from the group of: platelet rich plasma, laminin, collagen IV,heparin sulfate, entactin, and chondroitin sulfate. The term“extracellular matrix compound” is a term well-known to those skilled inthe art. Non-limiting examples of extracellular matrix compounds arecollagen, laminin, elastin, fibronectin, and the like, which are widelycommercially available. Substitutes thereof are also known in the artand are commercially available. Methods and protocols to prepareextracellular matrix compounds are also known in the art. Any home-madeor commercially available extracellular compounds can be used in thecomposition of the invention. In a preferred embodiment, theextracellular matrix compound is platelet rich plasma. Anyphysiologically acceptable concentrations (ml/ml) of platelet richplasma can be used in the present invention but a concentration in therange of about 0.0005-5 ml/ml, more preferably about 0.005-0.5 ml/ml,more preferably about 0.01-0.25 ml/ml, more preferably about 0.02-0.15ml/ml, more preferably about 0.03-0.10 ml/ml, more preferably about0.04-0.08 ml/ml, more preferably about 0.045-0.06 ml/ml, more preferablyabout 0.05 ml/ml of platelet rich plasma is preferred. In oneembodiment, the platelet rich plasma is derived from the peripheralblood of a donor subject, preferably a human subject. Protocols andmethods to obtain platelet rich plasma from the blood of a subject areknown in the art.

In an embodiment, at least two, three, four, five, six, or more,extracellular matrix compounds selected from the group of: platelet richplasma, laminin, collagen IV, heparin sulfate, entactin, and chondroitinsulfate, are present in the composition of the invention. It was foundby the present inventor that the incorporation of at least two,preferably three, preferably four, preferably five, more preferably sixextracellular matrix compounds into the composition of the invention,led to an increase in the effectiveness of the composition of theinvention, relative to composition wherein only one extracellular matrixcompound is present.

In another embodiment, the composition of the invention may optionallyfurther comprise one or more ingredients selected from the group of:albumin, essential and non-essential amino acids, vitamins, traceelements, organic constituents, inorganic salts, growth supplement, andantibiotics. It may be advantageous to add one or more of saidingredients to further promote growth and proliferation of hairfollicular stem cells while circumventing bacterial contamination in theculture medium.

In an embodiment, the composition taught herein does not comprise FoetalBovine Serum (also known as Foetal Calf Serum), or any animal serum,including human serum, unless the serum is autologous to the subject towhich a desired cell type or a desired tissue type is transplanted. Forexample, the desired cell type generated using the composition of theinvention may be transplanted into a subject, who may have also providedthe hair follicular stem cell.

In a highly suitable embodiment, the composition of the inventioncomprises Bis (maltolato) oxovanadium in a concentration in the range of0.01-100 mg/ml, more preferably 0.1-10 mg/ml, more preferably 0.5-8mg/ml, more preferably 0.6-6 mg/ml, more preferably 0.7-3 mg/ml, 0.8-2mg/ml, more preferably 0.9-1.5 mg/ml, more preferably about 1 mg/ml;erythropoietin in an amount of about 0.1 unit to 10 units/ml, preferablyabout 0.3 unit to 8 units/ml, more preferably about 0.5 unit to 6units/ml, even more preferably about 0.7 unit to 4 units/ml, yet morepreferably about 0.9 unit to 2 units/ml, even more preferably about 0.95unit to 1.5 units of erythropoietin/ml; at least one antioxidantcompound, preferably selected from the group of quercetin,monohydroxyethyl rutoside, vitamin C, deferoxamine mesylate, vitamin E,and lipoic acid, or any combination thereof, e.g., a combination ofmonohydroxyethyl rutoside, vitamin E and lipoic acid; at least oneextracellular matrix compound, e.g., platelet rich plasma; and at leastone differentiation inducing factor.

The differentiation inducing factor may be selected from the groupconsisting of: growth factors, such basic fibroblast growth factor,nerve growth factor, insulin-growth factor, epidermal growth factor,transforming growth factor, and epithelial growth factor; 5-azacytidine;activin A; taurin; and any combination thereof. Other non-limitingexamples of growth factors from which the growth factor may be selectedinclude: adrenomedullin, angiopoietin, autocrine motility factor, bonemorphogenetic proteins, brain-derived neurotrophic factor, glial cellline-derived neurotrophic factor, granulocyte colony-stimulating factor,granulocyte macrophage colony-stimulating factor, growth differentiationfactor-9, hepatocyte growth factor, hepatoma-derived growth factor,insulin-like growth factor, migration-stimulating factor, myostatin,neurotrophins, platelet-derived growth factor, transforming growthfactor alpha, transforming growth factor beta,tumor-necrosis-factor-alpha, vascular endothelial growth factor,placental growth factor, fetal bovine somatotrophin, and cytokines (e.g.IL-1-cofactor for IL-3 and IL-6, IL-2-t-cell growth factor, IL-3, IL-4,IL-5, IL-6, and IL-7). Any physiologically acceptable concentration(ng/ml) of growth factor may be used in the present invention but aconcentration in the range of about 0.01-100000 ng/ml, more preferablyabout 0.1-10000 ng/ml, more preferably about 1-1000 ng/ml, morepreferably about 10-500 ng/ml, more preferably about 50-400 ng/ml, morepreferably about 70-300 ng/ml, more preferably about 80-200 ng/ml, morepreferably about 90-150 ng/ml, more preferably about 95-125 ng/ml ofgrowth factor is preferred.

In an embodiment, the growth factor may be selected from one or more ofbasic fibroblast growth factor, insulin-like growth factor and nervegrowth factor, and the desired cell type and/or tissue type is neuralcell and/or neural tissue.

In an embodiment, the differentiation inducing factors used are thegrowth factors vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF), and the desired cell type is cells ofan endothelial lineage (Xu et al. Mol Med Rep. 2014, vol. 9:204-210).VEGF may be used in a contraction in the range of about 0.1-10,000ng/ml, more preferably about 1-1,000 ng/ml, more preferably about 10-500ng/ml, more preferably about 25-200 ng/ml, more preferably about 30-100ng/ml. bFGF may be used in a concentration of about 0.01-10,000 ng/ml,more preferably about 0.1-1,000 ng/ml, more preferably about 0.5-200ng/ml, more preferably about 1-100 ng/ml, more preferably about 5-50ng/ml.

In another embodiment, the differentiation inducing factors used aretransforming growth factor-β1 (TGF-β1) and platelet-derived growthfactor BB (PDGF-BB), and the desired cell type is contractile smoothmuscle cells (Xu et al. Mol Med Rep. 2013, vol. 8:1715-1721). TGF-β1 maybe employed in a concentration of about 0.01-10,000 ng/ml, morepreferably about 0.1-1,000 ng/ml, more preferably about 0.5-200 ng/ml,more preferably about 1-100 ng/ml, more preferably about 2-50 ng/ml.PDGF-BB may be employed in a concentration of about 0.01-10,000 ng/ml,more preferably about 0.1-1,000 ng/ml, more preferably about 0.5-200ng/ml, more preferably about 1-100 ng/ml, more preferably about 2-50ng/ml.

In one embodiment, the differentiation inducing factors are selectedfrom taurine, activin A and epidermal growth factor (EGF), or acombination thereof, and the desired cell type is a photoreceptor cell(cells expressing photoreceptor-specific markers rhodopsin, opsin andrecoverin), and/or tissue type is retinal tissue, e.g., retinal pigmentepithelium tissue (Kicic et al. 2003. J. Neurosci. Vol. 23:7742-7749).The obtained cells may be transplanted into the eye to counteract eyedegenerations, such as macular degeneration, e.g., age-related maculardegeneration. Taurin may be employed in a concentration in the range of0.1-1,000 μM, preferably 1-500 μM, more preferably 5-100 μM, mostpreferably 25-50 μM. Activin A may be employed in a concentration in therange of 0.1-10,000 ng/ml, preferably 1-1,000 ng/ml, more preferably5-500 ng/ml, yet more preferably 10-300 ng/ml. even more preferably50-200 ng/ml. Any physiologically acceptable concentration (ng/ml) ofeptidermal growth factor may be used but a concentration in the range ofabout 0.1-100,000 ng/ml, more preferably about 1-10,000 ng/ml, morepreferably about 10-1,000 ng/ml, more preferably about 50-500 ng/ml,more preferably about 60-400 ng/ml, more preferably about 70-300 ng/ml,more preferably about 80-200 ng/ml, more preferably about 90-150 ng/ml,more preferably about 95-125 ng/ml of epidermal growth factor ispreferred.

In one embodiment, the differentiation inducing factor is 5-azacytidineand the desired cell type is a cardiomyocyte (Potdar and Prasannan.2013. ISRN Stem cells. Article ID687282). The 5-azacytidine may beemployed in a concentration in the range of 0.1-1,000 μM, preferably1-500 μM, more preferably 5-100 μM, most preferably 5-50 μM.

In an embodiment, the growth factor is an epidermal growth factor andthe desired cell type and/or tissue type is selected from the groupconsisting of epidermal cell and/or epidermal tissue, skin cell and/orskin tissue, blood vessel cell and/or blood vessel tissue, and bone celland/or bone tissue. The epidermal growth factor may be selected from thegroup consisting of: heparin-binding epidermal growth-like growthfactor, epiregulin, epigen, betacellulin, neuregulin-1, neuregulin-2,neuregulin-3, and neuregulin-4.

In an embodiment, the growth factor is transforming growth factor, andthe desired cell type and/or tissue type is selected from the groupconsisting of cartilage cell and/or cartilage tissue, heart valve celland/or heart valve tissue, and skeletal muscle cell and/or skeletalmuscle tissue. The transforming growth factor may be selected from thegroup consisting of: transforming growth factor alpha and transforminggrowth factor beta.

In an embodiment, the growth factor is epithelial growth factor, and thedesired cell type and/or tissue type is keratinocyte cell and/orkeratinocyte tissue.

In an embodiment, the composition of the present invention furthercomprises a serum free growth medium. Serum free growth media arewell-known in the art and are readily commercially available. Theskilled person will know how to select an appropriate serum free growthmedium. In preferred embodiment, the serum free growth medium furthercomprises at least one ingredient selected from the group of: albumin,sodium chloride, potassium chloride, magnesium sulphate, sodiumphosphate, calcium chloride, glucose, sodium bicarbonate, sodiumlactate, sodium pyruvate, human serum albumin, and insulin.

In vivo method for generating a desired cell type and/or tissue typefrom at least one hair follicular stem cell.

Method of the Invention

In a second aspect, the present invention relates to an improved in vivomethod for generating a desired cell type and/or tissue type from atleast one hair follicular stem cell, said method comprising the stepsof:

(a) providing at least one hair follicular stem cell;(b) culturing the at least one hair follicular stem cell in a serum-freegrowth medium so as to obtain a population of hair follicular stemcells;(c) contacting the population of hair follicular stem cells of step (b)with a composition as taught herein and allowing said population of hairfollicular stem cells to differentiate into the desired cell type and/ortissue type under conditions favourable for differentiation; and(d) harvesting the desired cell type and/or tissue type.

In one embodiment, the at least one hair follicular stem cell of step(a) is obtained from at least a part of a hair follicle in the anagenphase. In another embodiment, the at least a part of a hair follicle inthe anagen phase has been obtained by plucking the hair from a donorarea of a subject. In a preferred embodiment, the donor subject ishuman. However, it is not essential that the hair follicular stem cellof step (a) be of human origin.

In step (a), the provision of at least a part of a hair follicle in theanagen phase can be performed by any methods known in the art, e.g.plucking one or more donor hairs from a donor subject, such as the scalpof a donor subject, and then selecting one or more donor hairs in theanagen phase. Selecting a donor hair in the anagen phase can beperformed by a person with ordinary skill in the art. It is well knownin the art that a hair in the anagen phase displays specificmorphological and histological characteristics that distinguish it froma hair in another phase of the growth cycle, such as the catagen phaseor telogen phase.

The hair may be plucked using any methods known in the art. Forexamples, the hair may be plucked using fingers or a plucking instrument(e.g. tweezers) may be used. In a embodiment, the donor hair is removedusing a plucking instrument such as a hollow harvesting needle, asdescribed in published international application WO2005077285.

In one embodiment, the at least a part of a hair follicle may becontacted with a medium comprising collagenase IV, in such a manner thatat least one hair follicular stem cell is enzymatically dissociated fromthe at least part of a hair follicle in the anagen phase. It may beparticularly advantageous to perform this step in a situation whereinthe at least one hair follicular stem cell of step (a) is still attachedor embedded within the hair follicle or part of a hair follicle. It isknown in the art that hair follicular stem cells can be enzymaticallydissociated from their respective hair follicle using enzymes such ascollagenases. Collagenases, particularly collagenase IV, are well-knownin the art and are commercially available. Protocols and methods fordissociating hair follicular stem cells using collagenases (e.g.collagenase IV) or other enzymes are also known and are used routinelyin the art.

In step (a), in certain cases, e.g. when a mixed population of hairfollicle cells is obtained from the hair follicle or part of a hairfollicle, it may be advantageous to isolate and purify the hairfollicular stem cells from the mixture, prior to conducting step (b).Methods and protocols for isolating, purifying, identifying, andseparating hair follicular stem cells are known in the art.

In an embodiment, the at least one hair follicular stem cell of step (a)may be cultured and expanded in a serum-free growth medium of step (b)for at least two weeks, e.g., at least 3 weeks. Serum-free growth mediaare known in the art and are commercially available. Protocols andmethods for preparing serum-free growth media are also known in the art.Any serum-free growth media can be used in the in vitro method of theinvention. In a preferred embodiment, the serum-free growth medium ofstep (b) is further supplemented with at least one tissue extract,preferably a bovine pituitary extract. Without wishing to be bound bytheory, it is hypothesized that the incorporation of a bovine pituitaryextract actively blocks the hair follicular stem cells from undergoingdifferentiation during the culture and expansion phase of step (b). ItIn an embodiment, step (b) may be repeated for an additional at leasttwo weeks, preferably at least 3 weeks in order to obtain an evengreater expansion of the population of hair follicular stem cells.

In step (b), the at least one hair follicular stem cell of step (a) iscultured in such a manner that a population of hair follicular stemcells is obtained.

In step (b), in certain cases, for instance in case of the occurrence ofcell death during the culturing period, it may be advantageous to removedead cell from the culture environment. Protocols and methods to removedead cells from a culture environment are known in the art. Forinstance, a gradient centrifugation method can be used to remove deadcells from the culture medium.

In step (b), it may be advantageous to replenish the culture medium ofstep regularly, preferably on a daily basis (i.e. every 24 hours).

In step (b), it may also be particularly advantageous to culture the atleast one hair follicular stem cell of step (a) in the context of athree-dimensional culture milieu. It is known in the art thatthree-dimensional cell culture systems allow various types ofinteractions to occur between the different cell types present in theculture in a manner that mimics what occurs in a natural environment.Methods and protocols for performing three-dimensional cell culture arealso well known in the art.

In an embodiment, the serum-free growth medium of step (b) may furthercomprise one or more ingredients selected from the group of albumin,essential and non-essential amino acids, vitamins, trace elements,organic constituents, inorganic salts, growth supplement, hormones,antibiotics, and tissue extracts. The addition of one or more of saidingredients may be advantageous to further promote proliferation,expansion, and growth of the hair follicular stem cells while preventingbacterial contamination or eliminating other pathogens in the culturemilieu.

In step (c), the population of hair follicular stem cells of step (b) iscontacted with a composition as taught herein; and said population ofhair follicular stem cells is allowed to differentiate into the desiredcell type and/or tissue type under conditions favourable fordifferentiation.

In an embodiment, the population of hair follicular stem cells of step(b) is contacted with the composition of step (c) for at least twoweeks, e.g., at least three weeks, at least four weeks, at least fiveweeks, at least six weeks, and the like. In an embodiment, the timeperiod during which the population of hair follicular stem cells of step(b) is contacted with the medium of step (c) varies depending on thedesired cell type and/or tissue type.

In another embodiment, the desired cell type of step (d) is selectedfrom the group consisting of: neural cell, photoreceptor cell such asretinal pigment epithelium cell and rods and cones, heart cell such ascardiomyocyte and pacemaker cell, tooth cell such as odontoblast andother pulp cells, epithelial cell, keratinocyte cell, fibroblast cell,fat cell, blood cell, immune cell, muscle cell, skin cell, hairfollicular stem cell, bone cell such as osteoblast, osteocyte,osteoclast, and cartilage cell such as chondrocyte. In a furtherembodiment, the desired tissue type of step (d) is selected from thegroup of: neural tissue, eye tissue, heart tissue, tooth tissue,epithelial tissue, keratinocyte tissue, connective tissue, fibroblasttissue, fat tissue, muscle tissue, skin tissue, hair tissue, bonetissue, and cartilage tissue.

In a preferred embodiment, the at least one hair follicular stem cell ofstep (a) is derived from a subject, and the composition of step (c)comprises at least one CD34-positive cell and/or platelet rich plasmaderived from said subject, and wherein the desired cell type and/ortissue type of step (d) is introduced in a recipient region in saidsubject. In a more preferred embodiment, the composition of step (c)comprises erythropoietin and/or platelet rich plasma derived from saidsubject, and wherein the desired cell type and/or tissue type of step(d) is introduced in a recipient region in said subject.

It may be particularly advantageous that the at least one hairfollicular stem cell of step (a) as well as one or more components ofthe composition of step (c) used to generate a desired cell type and/ortissue, are derived from the subject who will be receiving said newlygenerated desired cell type and/or tissue type. This is especiallyadvantageous in the context wherein an autologous cell and/or tissuetransplant into a subject is wanted. Overall, such procedure insuresthat (autologous) cell and/or tissue transplant or graft will be fullybiologically compatible with the recipient subject without theoccurrence of complications related to immune rejection and/or infectionin said subject.

In an embodiment, steps (a), (b), (c), and (d) are performed understerile conditions. Protocols and methods for manipulating, isolatingidentifying, purifying, culturing, maintaining, fertilizing, andharvesting cells, including hair follicular stem cells, are known in theart. It may be particularly advantageous to perform steps (a), (b), (c),and (d) under sterile conditions in the context where the new desiredcell type and/or tissue type obtained in step (d) is used in cosmeticprocedures in a subject, so as to avoid complications related to immunerejection and/or infection due to the presence of pathogens or otheragents capable of causing an infection or immune rejection.

Suitable Uses of the Method and Composition of the Invention

In a third aspect, the present invention relates to the use of a desiredcell type and/or tissue type obtainable or obtained by the in vitromethod for generating a new cell type and/or tissue type from at leastone hair follicular stem cell, as described herein, for a cosmeticand/or therapeutic purpose in a recipient subject. For instance, in oneembodiment, the desired cell type and/or tissue type obtainable orobtained by the in vitro method of the present invention is used forcosmetic or therapeutic tissue repair and/or cosmetic or therapeuticwound healing in a subject, e.g. subjects experiencing loss of tissue(e.g. skin, fat, muscle, tendon, etc.) due to cell and/or tissue damageinflicted by a wound, a burn, a tear, a scar, acne, wrinkles,degeneration, diseases and/or aging. In a further embodiment, thedesired cell type and/or tissue type obtainable or obtained by the invitro method of the present invention, is used for cosmetic facial andbody rejuvenation. For instance, the desired cell type and/or tissuetype obtainable or obtained by the in vitro method of the presentinvention can be used to cosmetically reshape a certain part of the faceand/or body of a subject, and/or to cosmetically modify (e.g. increase)the volume of a certain part of the face and/or body of a subject,and/or to cosmetically alter the texture of a certain part of the faceand/or body of a subject.

In an embodiment, the desired cell type and/or tissue type obtainable orobtained by the in vitro method for generating a new cell type and/ortissue type from at least one hair follicular stem cell, as describedherein, is used for therapeutic purposes, for instance regenerativemedicine, in a recipient subject in need thereof. In one embodiment thedesired cell type and/or tissue type obtainable or obtained by the invitro method of the present invention can be used to repair, regenerateor replace damaged tissues or organs as well as lost tissues or organs,which occur as a result of degeneration and/or necrosis and/or apoptosiscaused by aging, diseases, burns, accidents, injuries, cuts, ablationsand the likes.

All publications cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURE RELATED TO THE INVENTION

FIG. 1 displays the effect of the physiologically acceptable vanadiumcompounds oxovanadium (OVAN) and bis(maltolato) oxovanadium (BMOV) onproliferation of keratinocytes. Proliferation of keratinocytes isexpressed as the number of cells counted per amount of the culturemilieu (i.e. number of cells×10E4/ml). The results reveal an enhancedproliferation of keratinocytes following treatment with a compositioncomprising a basic medium growth serum (SFK) supplemented with 1 mg/mlof OVAN or 1 mg/ml of BMOV relative to the control situation (i.e. basicgrowth medium (SFK) lacking a vanadium compound). Note that treatmentwith BMOV is associated with a greater number of proliferated cellsrelative to treatment with OVAN.

EXAMPLES Example 1 Collection of Hair Follicular Stem Cells

Hair follicular stem cells were obtained from plucked hairs in theanagen phase, which were obtained from a donor subject, using a pluckinginstrument such as a hollow harvesting needle. Plucked hairs wereinspected under a microscope. Plucked hairs not displaying thecharacteristics of a hair in the anagen phase were discarded.

Example 2 Pre-Treatment of Hair Follicular Stem Cells

Plucked hairs in the anagen phase were immersed in 1% collagenase typeIV for 2 hours at 37° C. in order to enzymatically dissociate the hairfollicular stem cells from the hair follicle. Hair follicular stem cellswere then rinsed several times, and resuspended in culture medium.

Example 3 Collection and Culture of CD34-Positive Cells

CD34-positive cells act as “circulating fibrocytes” and their functionis dependent on the environment. For instance, in wound healingCD34-positive cells concentrate around the damaged tissue. In thecontext of tissue generation, CD34-positive cells act as a stem cellenhancer. Specifically, when hair follicular stem cells are culturedtogether with CD34-positive cells, hair follicular stem cells are ableto proliferate without supplement.

CD34-positive cells were obtained from the peripheral circulation of adonor subject. CD34-positive cells were isolated from the blood using aMACS cell separation kit (Miltenyl Biotec). Isolated CD34-positive cellswere then rinsed several time, resuspended in the culture medium (RPMI1640, GIBCO, Invitrogen) containing autologous serum, and cultured inthe same medium for a period of 8 weeks.

Example 4 Preparation of Culture Medium

The culture medium consisted of a sterile Serum Free Growth Medium(Defined Serum Free (Keratinocyte) Growth Medium, purchased from Gibco,USA) which was freshly prepared on the day of the experiment, accordingto the manufacturer's instructions.

Example 5 Preparation of the Composition for Generating a Desired CellType and/or Tissue Type

The composition for generating a desired cell type and/or tissue wasfreshly prepared by adding the following ingredients to the Serum FreeGrowth Medium described in example 4:

-   -   1 mg/ml of bis(maltolato)oxovanadium (BMOV)    -   0.25 mg/ml of mono Hydroxy-Ethyl Rutoside (mono-HER)    -   0.25 mg/ml of D-tocopherol acid succinate/α-tocopherol (vitamin        E)    -   0.1 mg/ml of lipoic acid    -   0.1 μmol/ml of adenosine triphosphate-magnesium chloride    -   15 mg/ml of deferoxamine mesylate    -   1×10³ cells/ml of CD34-positive cells    -   0.05 ml/ml of platelet rich plasma    -   1 unit/ml of Erythropoietin    -   Cell type and/or tissue-type specific differentiation inducing        factor: depending on the desired cell type and/or tissue type, a        specific additive is added. For instance, to generate neural        cell and/or tissue, a basic-fibroblast growth factor is added        (see example 6). To generate keratinocyte cell and/or tissue, an        epithelial growth factor is added (see example 7). To generate        neural cell and/or tissue, a neural growth factor is added (see        example 8).

Example 6 Generation of Keratinocyte Cell and/or Tissue

Hair follicular stem cells were enzymatically dissociated from the hairfollicular tissue obtained from 10 plucked hairs from a donor subjectusing collagenase IV (Example 2). Hair follicular stem cells werecultured and expanded for 3 weeks in a three-dimensional culture systemin the sterile Serum Free Growth Medium of Example 4. Subsequently, thepopulation of hair follicular stem cells was cultured in the compositionof Example 5 comprising epidermal growth factor (100 ng/ml) as thegrowth factor for a duration of 6 weeks. During this period, thecomposition comprising the epidermal growth factor was refreshed daily.At the term of the culture period, the keratinocyte cells were harvestedand submitted to immunohistological procedures using antibodies directedagainst keratinocyte markers, i.e. cytokeratins. Cytokeratins 1, 10 and11 are commonly used markers of differentiating keratinocytes and areexclusively found in the intermediate cells and in the granular cells atthe infundibulum in the outer root sheath (ORS) of the human anagen hairfollicles. Cytokeratins 19 is a marker of undifferentiated stem cells,and is found in outermost cells of the ORS at the isthmus and in somecells of the lower ORS. Cytokeratins 1, 10, 11, and 19 are used as areliable keratinocyte markers.

The results show that the newly produced keratinocyte cells and/ortissues were positive for cytokeratins 1, 10, 11, and 19 thusdemonstrating that keratinocyte cells and/or tissues can be generatedfrom hair follicular stem cells using the method and composition of thepresent invention.

Example 7 Generation of Nerve Cell and/or Tissue

Hair follicular stem cells were enzymatically dissociated from the hairfollicular tissue obtained from 10 plucked hairs from a donor subjectusing collagenase IV (Example 2). Hair follicular stem cells werecultured and expanded for 3 weeks in a three-dimensional culture systemin the sterile Serum Free Growth Medium of Example 4. Subsequently, thepopulation of hair follicular stem cells was cultured in the compositionset forth in Example 5 comprising nerve growth factor (100 ng/ml) as thegrowth factor for a duration of 6 weeks. During this period, thecomposition comprising the nerve growth factor was refreshed daily. Atthe term of the three weeks, nerve cells were harvested. At the term ofthe culture period, the nerve cells and/or tissues were harvested andsubmitted to immunohistological procedures using antibodies directedagainst neural markers, i.e. nestin. Another indication of a neural fateis the absence of the marker keratin 15. Therefore, cells and/or tissuespositive for nestin but negative for keratin 15 were reliably identifiedas neural cells and/or tissues.

The results show that the newly produced nerve cells and/or tissues werepositive for nestin and negative for keratin 15, thus demonstrating thatnerve cells and/or tissues can be generated from hair follicular stemcells using the method and composition of the present invention.

Example 8 Effect of Vanadium Compounds on Proliferation of Keratinocytes

Method:

Hair follicles were transferred to a 24-well culture disk containingdSFK with 500 mg/ml penicillin (Life Technologies B.V. Breda, TheNetherlands) and 0.25 μg/ml streptomycin (Life Technologies B.V. Breda,The Netherlands), and placed for 14 days in a culture medium at 31° C.in a humidified atmosphere containing 5% CO2. Three different culturemedium were used:

-   -   Treatment group oxovanadium (OVAN): The culture medium is a        composition comprising a basic SFK growth serum (purchased from        the supplier SFK, Enschede The Netherlands) supplemented with        either 1 mg/ml of OVAN.    -   Treatment group bis (maltolato) oxovanadium (BMOV): The culture        medium is a composition comprising a basic SFK growth serum        (purchased from the supplier SFK, Enschede The Netherlands)        supplemented with either 1 mg/ml of BMOV.    -   Control group: The control situation consists of a composition        comprising the SFK basic growth serum but without the vanadium        compounds (i.e. OVAN or BMOV).

The respective culture media were carefully removed every three days andreplaced by fresh culture media. The cells remained attached to the hairfollicles during this culture period. After 14 days the culture mediumwas removed and replaced by a 0.5 mg/ml trypsin, 0.2 mg/ml EDTA(ethylene diaminetetraacetic acid) solution (Life Technologies B.V.Breda, The Netherlands), and incubated for 5 minutes at 37° C. in thismedium. After this incubation period clusters of cells were releasedfrom the hair follicles. These were harvested by centrifugation at 300 gat 4° C. for 5 minutes in a Eppendorf 5804R Centrifuge (VWRInternational, The Netherlands). Number of cells were counted andexpressed as amount of proliferated cells per ml of culture medium.

Results:

The results show that the amount of proliferated keratinocytes wassignificantly increased following treatment with both vanadiumcompounds, i.e. OVAN and BMOV relative to the control situation. Theresults further show that BMOV appears to be more potent than OVAN.Overall, these results show that vanadium compounds are particularlyeffective at promoting cell proliferation as well as cell survival underculture condition (see FIG. 1).

1. A composition for generating a desired cell type and/or tissue typefrom at least one hair follicular stem cell, said composition comprisingat least one anti-apoptotic compound, wherein said anti-apoptotic agentis a physiologically acceptable vanadium compound, at least oneanti-oxidant compound, at least one stem cell enhancer compound, atleast one extracellular matrix compound, and at least onedifferentiation inducing factor.
 2. The composition according to claim1, wherein the physiologically acceptable vanadium compound is selectedfrom the group consisting of bis (maltolato) oxovanadium, oxovanadium,and orthovanadium.
 3. The composition according to claim 1, wherein thephysiologically acceptable vanadium compound is bis (maltolato)oxovanadium.
 4. The composition according to claim 1, further comprisingone or more second or further anti-apoptotic compounds selected from thegroup of a triiodothyronine, estradiol, progesterone, tissue extract,insulin, transferrin, selenium, L-cysteine, adenosinetriphosphate-magnesium chloride, and L-leucine.
 5. The compositionaccording to claim 4, wherein the tissue extract is bovine pituitaryextract.
 6. The composition according to claim 1, wherein theanti-oxidant compound is selected from the group of quercetin,monohydroxyethyl rutoside, vitamin C, lipoic acid, deferoxaminemesylate, and vitamin E.
 7. The composition according to claim 6,wherein the anti-oxidant compound is selected from quercetin andmono-hydroxyethyl rutoside.
 8. The composition according to claim 1,wherein the stem cell enhancer compound is selected from the group of:erythropoietin, CD34-positive cell, and retinoic acid.
 9. Thecomposition according to claim 8, wherein the stem cell enhancercompound is selected from the group of erythropoietin and CD34-positivecell.
 10. The composition according to claim 8, wherein the stem cellenhancer compound is erythropoietin.
 11. The composition according toclaim 8, wherein erythropoietin and/or the CD34-positive cell is derivedfrom peripheral blood or bone marrow of a donor subject.
 12. Thecomposition according to claim 8, wherein the CD34-positive cell isobtainable or obtained from a CD34-positive cell line.
 13. Thecomposition according to claim 8, wherein the CD34-positive cell isobtainable or obtained from a human CD34-positive cell line.
 14. Thecomposition according to claim 1, further comprising at least onedegranulating agent.
 15. The composition according to claim 14, whereinthe degranulating agent is Compound 48/80.
 16. The composition accordingto claim 14, further comprising an inorganic salt.
 17. The compositionaccording to claim 16, wherein the inorganic salt is CaCl₂.
 18. Thecomposition according to claim 1, wherein the extracellular matrixcompound is selected from the group of: platelet rich plasma, laminin,collagen IV, heparin sulfate, entactin, and chondroitin sulfate.
 19. Thecomposition according to claim 18, wherein the extracellular matrixcompound is platelet rich plasma.
 20. The composition according to claim19, wherein the platelet rich plasma is derived from peripheral blood ofa donor subject.
 21. The composition according to claim 1, furthercomprising one or more ingredients selected from the group of albumin,essential and non-essential amino acids, vitamins, trace elements,organic constituents, growth supplement, and antibiotics.
 22. Thecomposition according to claim 1, wherein the desired cell type isselected from the group of nerve cell, photoreceptor cell,cardiomyocyte, odontoblast, epithelial cell, keratinocyte cell,fibroblast cell, fat cell, blood cell, immune cell, muscle cell, skincell, hair follicular cell, osteoblast, osteocyte, osteoclast, andchondrocyte.
 23. The composition according to claim 1, wherein thedesired tissue type is selected from the group of neural tissue, eyetissue, heart tissue, tooth tissue, epithelial tissue, keratinocytetissue, fibroblast tissue, connective tissue, fat tissue, muscle tissue,skin tissue, hair tissue, bone tissue, and cartilage tissue.
 24. Thecomposition according to claim 1, wherein the differentiation inducingfactor is selected from the group consisting of basic fibroblast growthfactor, insulin-growth factor, epidermal growth factor, transforminggrowth factor, nerve growth factor, fibroblast growth factor, epithelialgrowth factor, taurin, activin A, and 5-azacytidine.
 25. The compositionaccording to claim 1, wherein the differentiation inducing factor isselected from basic fibroblast growth factor, insulin-growth factor, andnerve growth factor, and said desired cell type and/or tissue type isneural cell and/or neural tissue.
 26. The composition according to claim25, wherein said insulin-like growth factor is selected from the groupconsisting of insulin-like growth factor type 1 and insulin-like growthfactor type
 2. 27. The composition according to claim 1, wherein thedifferentiation inducing factor is epidermal growth factor, and saiddesired cell type and/or tissue type is epidermal cell and/or tissuetype, skin cell and/or tissue type, blood vessel cell and/or tissuetype, or bone cell and/or tissue type.
 28. The composition according toclaim 27, wherein the epidermal growth factor is selected from the groupconsisting of: heparin-binding epidermal growth-like growth factor,epiregulin, epigen, betacellulin, neuregulin-1, neuregulin-2,neuregulin-3, and neuregulin-4.
 29. The composition according to claim1, wherein the differentiation inducing factor is transforming growthfactor, and said desired cell type and/or tissue type is cartilage celland/or tissue type, heart valve cell and/or tissue type, and skeletalmuscle cell and/or tissue type.
 30. The composition according to claim29, wherein the transforming growth factor is selected from the groupconsisting of: transforming growth factor alpha and transforming growthfactor beta.
 31. The composition according to claim 1, wherein thedifferentiation inducing factor is selected from activin A, taurin, andepidermal growth factor, and said desired cell type and/or tissue typeis photoreceptor cell and/or retinal tissue.
 32. The compositionaccording to claim 1, wherein the differentiation inducing factor isfibroblast growth factor, and said desired cell type and/or tissue typeis fibroblast cell, skin cell and/or skin tissue, or blood vessel celland/or blood vessel tissue.
 33. The composition according to claim 1,wherein the differentiation inducing factor is epithelial growth factor,and said desired cell type and/or tissue type is keratinocyte celland/or keratinocyte tissue.
 34. The composition according to claim 1,which further comprises a serum free growth medium.
 35. An in vitromethod for generating a desired cell type and/or tissue type from atleast one hair follicular stem cell, said method comprising the stepsof: (a) providing at least one hair follicular stem cell; (b) culturingthe at least one hair follicular stem cell in a medium so as to obtain apopulation of hair follicular stem cells; (c) contacting the populationof hair follicular stem cells of step (b) with a composition accordingto claim 1 and allowing said population of hair follicular stem cells todifferentiate into the desired cell type and/or tissue type underconditions favorable for differentiation; and (d) harvesting the desiredcell type and/or tissue type.
 36. The in vitro method according to claim35, wherein the at least one hair follicular stem cell of step (a) isobtained from at least a part of a hair follicle in the anagen phase.37. The in vitro method according to claim 36, wherein the at least apart of a hair follicle in the anagen phase has been obtained byplucking a hair from a donor area of a subject.
 38. The in vitro methodaccording to claim 35, wherein the at least a part of a hair follicle iscontacted with a medium comprising collagenase IV.
 39. The in vitromethod according to claim 35, wherein the hair follicular stem cell is ahuman hair follicular stem cell.
 40. The in vitro method according toclaim 35, wherein the medium is a serum-free growth medium, optionallyfurther supplemented with at least one tissue extract.
 41. The in vitromethod according to claim 40, wherein the tissue extract is preferably abovine pituitary extract compound.
 42. The in vitro method according toclaim 35, which desired cell type and/or tissue type is intended forintroduction in a recipient region in a subject, and wherein the atleast one hair follicular stem cell of step (a) is derived from saidsubject, and wherein the composition of step (c) comprises at least oneCD34-positive cell derived from said subject and/or platelet rich plasmaderived from said subject.
 43. Use of a desired cell type and/or tissuetype obtainable or obtained by the in vitro method according to claim35, for a cosmetic purpose.
 44. Use of a desired cell type and/or tissuetype obtainable by the in vitro method according to claim 35, forcosmetic tissue repair and/or wound healing.
 45. Use of a desired celltype and/or tissue type obtainable by the in vitro method according toclaim 35, for cosmetic facial and body rejuvenation.
 46. Use of adesired cell type and/or tissue type obtainable by the in vitro methodaccording to claim 35 for regenerative medicine.
 47. A kit comprisingthe composition according to claim
 1. 48. The kit according to claim 46further comprising information on how to use the composition to generatea desired cell and/or tissue type.